Enzymatic determinations in Calcofluor-resistant (cal1) mutants of Saccharomyces cerevisiae as well as cloning and sequencing of the CAL1 gene indicated that those mutants lack the newly discovered chitin synthetase 3 (Chs3). Examination of strains lacking either chitin synthetase 2 (Chs2) or Chs3 by electron microscopy and specific staining of chitin indicated that Chs3 is responsible for the chitin ring formed at the base of emerging buds and for chitin dispersed in the cell wall, whereas Chs2 is required for the chitin disk synthesized between mother and daughter cells at cytokinesis. Chs3 is also involved in the diffuse deposition of chitin in cell cycle mutants incubated at a nonpermissive temperature. Finally, Chs3 is required for the formation of a chitosan layer in spore walls. Simultaneous deficiency in Chs2 and Chs3 is lethal, therefore an inhibitor of both Chs2 and Chs3 may be an effective antifungal agent.